Biological effects of Lippia alba essential oil against Anopheles gambiae and Aedes aegypti

The management of mosquito resistance to chemical insecticides and the biting behaviour of some species are motivating the search for complementary and/or alternative control methods. The use of plants is increasingly considered as a sustainable biological solution for vector control. The aim of this study was to evaluate the biological effects of the essential oil (EO) of Lippia alba harvested in Abidjan (Côte d’Ivoire) against Anopheles gambiae and Aedes aegypti mosquitoes. Phytochemical compounds were identified by GC–MS. Knockdown and mortality were determined according to the WHO test tube protocol. Contact irritancy was assessed by observing the movement of mosquitoes from a treated WHO tube to a second untreated tube. Non-contact repellency was assessed using a standardised high-throughput screening system (HITSS). Blood meal inhibition was assessed using a membrane feeding assay treated with EO. The EO was identified as the citral chemotype. The EO gave 100% KD60 in both species at a concentration of 1%. Mortalities of 100% were recorded with An. gambiae and Ae. aegypti at concentrations of 1% and 5% respectively. The highest proportions of females escaping during the contact irritancy test were 100% for An. gambiae at 1% concentration and 94% for Ae. aegypti at 2.5% concentration. The 1% concentration produced the highest proportions of repelled mosquitoes in the non-contact repellency tests: 76.8% (An. gambiae) and 68.5% (Ae. aegypti). The blood meal inhibition rate at a dose of 10% was 98.4% in Ae. aegypti but only 15.5% in An. gambiae. The citral chemotype of L. alba EO has promising biological effects in both species that make it a potentially good candidate for its use in mosquito control. The results obtained in this study encourage the further evaluation of L. alba EOs from other localities and of different chemotypes, under laboratory and field conditions.


Mosquitoes
Bioassays were carried out on mosquitoes of the genus Aedes and Anopheles susceptible to chemical insecticides and maintained at the IRD insectarium (27 ± 1 °C and 70 ± 10% relative humidity) in Montpellier: Kisumu strain of An. gambiae from Kenya and SBE strain of Ae. aegypti from Benin.The photoperiod was set at 14 h light and 10 h dark.

GC-MS data acquisition
GC-MS was used to identify the essential oil compounds.1 µL of a 2% solution of the essential oil diluted in hexane was injected into the Agilent 8860 System chromatograph coupled to the Agilent 5977B GC/MSD mass spectrometer.The oven temperature was initially set at 50 °C for 2 min, before being increased to 110 °C (4 °C/min), 250 °C (8 °C/min) and 310 °C (10 °C/min).The carrier gas was helium at a temperature of 250 °C, a pressure of 7.6522 psi and a total flow rate of 104 mL/min in split mode.The Agilent 122-5032 column (30 m × 250 μm × 0.25 μm) had a constant flow rate of 1 mL/min.The temperature of the mass detector was 300 °C.

Data preprocessing of the GC-MS data
GC-MS data were first analyzed and processed using MzMine 2.53 to visualize the chromatograms and spectra 47,48 .Then, GC-MS raw data were converted to Analysis Base File (ABF) format using ABF converter (http:// www.reify cs.com/ AbfCo nvert er/ index.html).Next, the converted data were pre-processed using MS-DIAL 4.70 (NSF-JST, Japan), including peak extraction, peak alignment, baseline calibration, deconvolution analysis and peak identification.We selected KovatsRI based on alkanes as a retention index for peak alignment.The deconvoluted spectra in NIST MSP format (GC-MS DB-Public-KovatsRI-VS3) were imported and matched with spectral libraries.Those peaks with an average peak width of 20 scans and minimum peak height above 10,000 amplitudes were selected for peak detection.Peaks with a s-window value of 0.5 and EI spectral cutoff of 5000 amplitudes underwent a deconvolution operation.The identification parameters were set as follows: m/z tolerance of 0.5 Da, retention time tolerance of 0.5 min, EI similarity cutoff value of 70% and identification score cutoff value of 70%.The alignment parameters of retention time tolerance and retention time factor were set to 0.075 min and 0.5, respectively.

Adulticidal tests
The insecticidal activity of the EO of L. alba was evaluated at dilutions of 0.1%, 1%, 2.5% and 5% (v/v).Permethrin (91.8% technical grade) was used as a positive control (0.011 and 0.11 mg/cm 2 ).The EO was diluted in a solution of ethanol plus silicone oil Dow Corning 566 (134 mL ethanol + 66 mL silicone) and permethrin in a solution of acetone plus silicone oil Dow Corning 566 (134 mL acetone + 66 mL silicone).Two milliliters of the different solutions were used to impregnate the filter papers (12 × 15 cm, Whatman®).The tests were conducted with slight modifications to the protocol of Deletre et al. 21, at 27 ± 2 °C and 80 ± 10% relative humidity.Two WHO tubes were used, one marked with a red dot for mosquito exposure to impregnated paper and one marked with a green dot containing unimpregnated paper for mosquito holding.A total of 25-30 females aged 3-7 days (postemergence) were introduced into three holding tubes.After 30 min of acclimatation, the dead mosquitoes, if any, were replaced.The mosquitoes were then transferred to the exposure tubes for 60 min.After exposure time, knocked down mosquitoes (KD60) were counted and all the mosquitoes were transferred back to the holding tubes.Tubes were kept in a climatic chamber (27 ± 2 °C and 80 ± 10% relative humidity) for 24 h.During this time, individuals were provided with a cotton soaked with a 10% honey solution for feeding.The number of dead mosquitoes were checked at 24 h after exposure.For each product and concentration, mosquitoes exposed to papers impregnated with the solvent only served as negative controls.Mortality for treated mosquitoes was corrected by the Abbott formula when mortality in control mosquitoes was between 5 and 20%: (%treatment mortality−%control mortality)/(100−control mortality) × 100.

Contact irritancy tests
The contact irritancy of the EO was evaluated at concentrations of 0.1%, 1%, 2.5% and 5%.The tests were performed in WHO tubes.Two tubes (one marked with a red dot and the other with a green dot) were connected with a slide door unit and placed horizontally on the laboratory bench.Whatman® paper (12 × 15 cm) impregnated with the test solution (2 mL) was placed in the red-dotted tube and unimpregnated paper in the green-dotted tube.The papers were held against the walls of the tubes with metal clips.The device for the negative control consisted of a tube containing a solvent impregnated paper (ethanol and silicone for the essential oil, acetone and silicone for permethrin) and a tube containing unimpregnated paper.Twenty to twenty-four (20-24)  females aged 3-7 days after adult emergence were introduced into the tube containing the impregnated paper.After 30 s of acclimatation, the slide unit door was opened and closed after 10 min.Mosquitoes were then killed in a freezer.Mosquitoes present in each tube were then counted and the proportion of passage from treated to untreated tube was determined 21,49 .Permethrin was evaluated as a positive control.The test was validated when the proportion of escaped mosquitoes in the negative controls was less than 50%.The test was performed three times for each dose.Tests were performed at 27 ± 2 °C and 80 ± 10% relative humidity.

Non-contact repellency tests
The repellent effect of the EO of L. alba was evaluated using a modified High-Throughput Screening System inspired by the methodology proposed in Grieco et al. 50and Deletre et al. 21.The system consisted of two cylindrical chambers (treated and untreated) connected by a door.Whatman paper (10 × 30 cm) impregnated with the solution to be tested (3.3 mL) was placed between a rigid transparent film and a mosquito net.The assembly was rolled around a drum so that the transparent film was on the exterior of the armature and the whole was placed in the treated chamber.Unimpregnated paper was placed in the untreated chamber.The treated chamber for the control mosquitoes contained paper impregnated only with solvent (ethanol-silicone oil Dow Corning 566).Twenty to twenty-four fasting females aged 3-7 days (post-emergence) were introduced into the treated chamber.The door was opened after 30 s of acclimatation and closed again after 10 min (Fig. 1).Mosquitoes in each of the chambers were anesthetized with CO 2 and then counted.The proportion of passage from the treated to the untreated chamber was then determined.The essential oil was evaluated at 0.1, 1, 2.5 and 5%.The test was performed three times for each dose and was validated when the proportion of escaped mosquitoes in the negative controls was less than 50%.Bioassays were carried out at 27 ± 2 °C and 80 ± 10% relative humidity.

Blood feeding inhibition
Solutions of L. alba EO at 1%, 2.5%, 5% and 10% were prepared with absolute ethanol (v/v).The first three solutions were evaluated before the 10% essential oil solution and ethanol was used as a negative control for each test.Glass feeders (Ø = 16.32 mm) were connected to each other by silicon tubing: 12 feeders for the evaluation of the first 3 solutions or 6 feeders for the evaluation of the 10% solution.They were then connected to a water bath equipped with a pump to circulate water through the feeders at 37 °C.The feeders were positioned on the racks at a rate of three feeders per dose.Pre-cut pig gut membranes were placed on the underside of the feeders.Ten (10) µL of the EO solutions were spread on each membrane.The feeders were then turned upside down and 100 µL of rabbit blood was introduced into each feeder.Cups (440 mL) covered with mosquito netting containing 20-29 adult females aged 5-10 days old were placed under each feeder.After one hour, the mosquitoes were removed and stunned in the freezer before counting the blood fed mosquitoes (Fig. 2).The test was validated when the feeding rate in control mosquitoes was at least 75%.The blood meal inhibition rate was defined as: [1−(% fed EO/% fed Ethanol)] × 100.The tests were carried out in 3 replicates at 27 ± 2 °C and 80 ± 10% relative humidity.

Statistical analysis
The data were analysed using R-4.2.1 software 51 .The proportions of KD60, dead or displaced mosquitoes induced by the essential oil were compared to those induced by the different negative controls using Fisher exact test.
The generalized linear mixed model with binomial distribution was used for the analysis of the blood meal

Adulticidal tests
The 0.1% EO produced very low KD60 and mortality An. gambiae individuals (7.8% and 4.5% respectively).The mortality recorded at this concentration was not significantly different from the negative control.From the 1% concentration onwards the KD60 and mortality rates were 100% except for the 2.5% which resulted in 97.6% mortality.Permethrin caused 100% KD60 and mortality at 0.011 and 0.11 mg/cm 2 (Table 2).Contrary to the other concentrations, the KD60 and mortality induced by 0.1% EO on Ae. aegypti were not significantly different from the negative control.At 1%, KD60 was 100% but mortality was only 16.4%.The KD60 and mortality rates recorded with the 2.5% solution were 100% and 64.2% respectively.The 5% dose induced 100% KD60 and mortality.Permethrin caused 100% KD60 and mortality at 0.011 and 0.11 mg/cm 2 (Table 2).

Contact irritancy tests
The proportions of An. gambiae females escaping from the EO-treated tubes to the untreated tubes were 60.8% and 100% at the 0.1 and 1% doses respectively.These proportions decreased to 79.6% for the 2.5% concentration and 70.4% for the 5% concentration.At concentrations of 0.011 and 0.11 mg/cm 2 , permethrin caused 23.1 and 48.5% displacement of mosquitoes to untreated tubes, respectively (Table 3).
The mean rates of blood fed Ae. aegypti significantly decreased as concentrations increase (Table 4).The blood feeding inhibition rates increases from 28.4% for the 1% concentration to 98.5% for the 10% concentration (Fig. 4).

Discussion
The management of mosquito resistance to chemical insecticides and the biting behaviour of some species are motivating the search for complementary and/or alternative control methods.We identified the molecules present in the essential oil of L. alba and determined its biological effects (insecticidal, repellent and blood meal inhibition) on An. gambiae and Ae.aegypti.Mainly composed of citral, the EO has been shown to be toxic, irritant and repellent against both species of mosquito.Unexpectedly, the lowest blood meal inhibition rate was recorded with the highest dose of EO with An. gambiae, while this was not observed with Ae. aegypti.These results provide the basis for future work on the impact of L. alba EO formulations for protection against mosquito bites in the field.
The EO evaluated in this study consists mainly of citral (45.86%, geranial and neral being the two isomers of citral) and geraniol (14%).The chemotypes of L. alba EOs, which can be associated with the genetic forms of the plant species, have been characterised according to their main compounds, the variability of which can    29 .The insecticidal activity of some EOs may be related to the inhibition of mitochondrial enzymes and acetylcholinesterase (AChE) activities by their compounds 60,61 .In this study, the EO of L. alba has been shown to be toxic to insecticide susceptible strains of mosquitoes, as it induced 100% KD60 in An. gambiae and Ae.aegypti at doses as low as 1%.Dua et al. 18 also obtained 100% KD60 with the EO of L. camara another Verbenaceae mainly composed of caryophyllene, eucalyptol, α-humelene and germacrene-D, at a dose of 4% with Anopheles culicifacies, Anopheles fluviatilis, Anopheles stephensi, Cx. quinquefasciatus and Ae.aegypti.The carvone chemotype of L. alba EO at 0.1% induced 80% mortality in Ae. aegypti adults 39 , whereas the citral chemotype evaluated in the present study induced 4.5% mortality in An. gambiae and 2.3% in Ae. aegypti at the same dose.This divergence in mortality can be explained either by the higher insecticidal effect of carvone chemotype or by the use of the CDC bottle by Castillo et al. 39 and the fact that EO deposited on glass may be more effective than on paper that could adsorb a part of the EO.Significant mortalities were observed in this study from 1% with An. gambiae and 2.5% with Ae. aegypti.Larvicidal activity of several chemotypes of L. alba EO against Ae.aegypti, Ae. albopictus and Cx.quinquefasciatus has also been reported 40,[62][63][64] .Moreover, the carvone and citral chemotypes of L. alba have been shown to be toxic against other insects (Ulomoides dermestoides, Sitophilus zeamais and Tribolium castaneum) 65,66 and Rhipicephalus (Boophilus) microplus tick 67,68 .
The 1% solution of L. alba EO gave 100% KD60 but only 16.4% mortality with Ae. aegypti.These results have also been observed when using intermediate concentrations of EOs as for instance 3-5% solutions of C. nardus, N. cataria and O. americanum which caused 100% KD60 and less than 10% mortality on Ae. aegypti 69 .The recovery of Ae. aegypti females after knockdown may be explained by natural detoxification mechanisms or by reversible inhibition of receptors in the nervous system as observed with some insecticides, including natural pyrethrins 70,71 .
In the present study, the 1% concentration induced 76.8 and 68.5% repellency on An. gambiae and Ae.aegypti respectively.The 2.5 and 5% concentrations gave lower repellent rates than those obtained with 1% on both species.As the tests were carried out in hermetically closed cylinders, the 2.5 and 5% concentrations could have started to intoxicate the mosquitoes or impaired their sensory systems.Cutaneous application of the citral chemotype of L. alba EO protects against mosquito bites 41 .The EO of L. alba, composed mainly of 2, 7 octadione-1-butoxy and 2-isopropenyl-5-methyl hex-4-enol, has a repellent activity, unlike that composed mainly of carvone and limonene 39,40 .Odour Binding Proteins (OBPs) transport volatile molecules to olfactory receptors which reside in contact with the dendrites of sensory neurons.Citral and limonene showed good docking with OBP 1 and OBP 22 , which have been identified as mediators of olfaction in Ae. aegypti, in addition to acetylcholinesterase 72,73 .One of the potential mechanisms of action of the repellent compounds in EOs such as Lippia thymoides, Cymbopogon winterianus, Eucalyptus globulus and L. alba is their interaction with OBP 1 41 .The irritant effect observed in the contact repellency test may not only be as a consequence of the intrinsic irritancy of the EO, since a spatial repellency effect was observed for the two mosquito species.In fact, the movement of mosquitoes away from a treated surface without and after contact involves the binding of molecules to specific olfactory receptors on the antennae and specific gustatory receptors on the tarsi, respectively 74 .These two mechanisms would therefore have resulted in the escape of An. gambiae and Ae.aegypti females from tubes treated with L. alba EO to untreated tubes during contact irritancy tests.This irritancy increased from 60.8 to 100% from 0.1 to 1% concentrations, but fell to 79.6% and 70.4% respectively at 2.5% and 5% on An. gambiae.In Ae. aegypti, the proportion of escaped females was 77.4% with the 5% solution, compared to 94.1% with 2.5%.As the EO showed a neurotoxic effect, the apparent reduction of irritancy are likely to be due to some mosquitoes starting to be intoxicated or being knocked down during the 10 min the slide unit was opened.Reduced irritability caused by a knock down effect has already been observed with DEET and IR3535 against Ae.aegypti 75 .
The proportions of blood fed An. gambiae ranged from 34 to 49% with 1-5% concentrations of L. alba EO.Wangrawa et al. 76 obtained lower proportions of blood fed females using tunnel tests: 20.5% with 1.5% L. camara, 30.3% with 3% Ocimum canum, 35.3% with 2.5% Hyptis spicigera and 19.5% with 2% Hyptis suaveolens.This may be due to the fact that tunnel tests not only measure the direct blood feeding inhibition but also the combined effects of mortality and excito-repellency when mosquitoes are exposed to a treated netting.The highest rate of blood fed females (76.6%) was observed with the 10% concentration of L. alba EO on An. gambiae in the present study.This unexpected observation has already been reported by Hodjati and Curtis 77 , who obtained higher feeding rates with the highest dose of permethrin on susceptible and resistant populations of An. stephensi.This high dose was also associated with the lowest KD and mortality in their study.L. alba EO had a better effect on blood meal inhibition of Ae. aegypti with an inhibition rate of 98.4% for the 10% concentration.The behavioural differences found in both species as well as the unexpectedly high blood feeding rate of An. gambiae at high concentration will necessitate further investigations.

Conclusion
The investigations carried out showed that the EO of L. alba had very promising knockdown, toxicity, irritant and repellent effects as well as a blood feeding inhibition on An. gambiae and Ae.aegyti.It is clearly a good candidate for the development of sustainable biological products for mosquito control.To this end, further evaluations under field conditions should be carried out to measure the effect of the extract on mosquito bites and/or pathogen transmission.The results obtained have prompted also an evaluation of the biological effects of EOs of L. alba harvested in different localities of west Africa against mosquito vectors of pathogens.Finally, it will be interesting to investigate the impact high doses of L. alba EO on odorant receptors of An. gambiae and their consequences on mosquito behaviour. https://doi.org/10.1038/s41598-024-52801-1www.nature.com/scientificreports/

Table 2 .
Mortality and KD60 (Knockdown at 60 min) of L. alba essential oil (EO) and permethrin against An.
Vol.:(0123456789) Scientific Reports | (2024) 14:3508 | https://doi.org/10.1038/s41598-024-52801-1www.nature.com/scientificreports/be related to the morphological characteristics of the leaves and the localities or environmental conditions of production 29,53-59 .Hennebelle et al. 53 classified the EOs of L. alba into seven chemotypes on the basis of the phytochemical profiles of 4 samples from the French overseas departments and those of 54 other samples reported in 35 publications.This classification can be summarised as follows: (I) citral, linalool, caryophyllene as the major compounds (four subtypes); (II) tagetenone; (III) limonene, carvone in variable amounts, often replaced Table 1.Phytochemical composition of the essential oil of L. alba leaves harvested in Abidjan (Côte d'Ivoire).Rt, retention time.

Table 3 .
Contact irritancy and repellent effects of L. alba essential oil (EO) on An. gambiae and Ae.aegypti (permethrin = positive control of contact irritancy tests).SBE, sensitive strain of Ae. aegypti from Benin; n, number of mosquitoes exposed, 95% CI: 95% confidence interval.The proportions of displaced mosquitoes induced by the essential oil were compared to those induced by the different negative controls using Fisher exact test; *** :P < 0.0001, **: P < 0.001, *: P < 0.05.